Sendai virus is also named hemagglutinating virus of Japan (HVJ), and classified in parainfluenza virus type I, belonging to the genus Paramyxovirus of the family Paramyxoviridae.
Sendai virus particle is pleomorphic, having the genome RNA without a function as template for translation (hereafter designated “negative strand RNA”) enclosed in an envelope of 150-200 nm in diameter. Historically, Sendai virus has also been regarded as an industrially useful virus, being widely utilized, especially for the production of heterokaryons and hybrid cells, by taking advantage of viral cell-fusion capacity. Also, cell-fusing liposomes have been developed as a vehicle for gene therapy. Furthermore, Sendai virus is also used as the inducer for various interferons.
According to the classification based on the karyotype of genome nucleic acid, Sendai virus belongs to a group of negative single-strand RNA viruses of negative strand RNA viruses among RNA viruses. RNA viruses are classified into three groups, the dsRNA viruses (double stranded RNA viruses), positive strand RNA viruses, and negative strand RNA viruses. The ds RNA virus group includes reovirus, rotavirus, phytoreovirus, etc., and have segmented, plural filamentous dsRNA genome. Positive strand RNA viruses include poliovirus, Sindbis virus, Semliki forest virus, and Japanese B encephalitis virus, which possess a single positive sense RNA as genome. The genome RNA can function as an mRNA and is capable of producing proteins required for RNA replication and particle formation depending on the translational functions of host cells. In other words, the genome RNA itself of positive strand RNA viruses is capable of disseminating. In the present specification, by “disseminative capability” is meant “the capability to form infectious particles or their equivalent complexes and successively disseminate them to other cells following the transfer of nucleic acid into host cells by infection or artificial techniques and the intracellular replication of said nucleic acid. Sindbis virus classified to positive strand RNA viruses and Sendai virus classified to negative strand RNA viruses have both infectivity and disseminative capability. On the other hand, adeno-associated virus classified to the parvovirus family has the infectivity but no disseminative capability (the mixed infection of adenovirus is necessary for the formation of viral particles). Furthermore, the positive strand RNA derived from Sindbis virus which is artificially transcribed in vitro is disseminative (to form infectious viral particles when transfected into cells). In contrast, not only negative strand but also positive strand of Sendai viral RNA artificially transcribed in vitro is not disseminative (form no infectious viral particles when transfected into cells).
Recently, viral vectors have been used as vehicles for gene therapy. In order to use them as gene therapy vectors, it is necessary to establish techniques for reconstituting viral particles. (By “reconstitution of viral particles” is meant the artificial formation of viral genome nucleic acid and the production of original or recombinant viruses in vitro or intracellularly.) This is because, in order to transfer foreign genes into viral vectors, viral particles should be reconstituted from the viral genome with foreign genes integrated by the gene manipulation. Once techniques of viral reconstitution are established, it becomes possible to produce viruses with a desired foreign gene introduced, or with desired genes deleted or inactivated.
Also, once the viral reconstitution system is constructed and the viral gene manipulation becomes possible, said system appears to become a potential tool for genetically analyzing the viral function. Genetic analysis of viral functions is very important from the medical viewpoint of prevention and therapy of diseases etc. For example, if the replication mechanism of viral nucleic acid is elucidated by utilizing the differences between viral metabolism and host-cellular metabolism, it may be possible to develop viricide acting on the viral nucleic replication process and less damaging to host cells. Also, by elucidating functions of viral gene-encoded proteins, it may become possible to develop antiviral drugs targeting proteins related with the viral infectivity and particle formation. Furthermore, by modifying genes concerned with the membrane fusion and preparing liposomes with superior membrane-fusing capability, it will be able to use them as gene therapy vectors. In addition, as represented by the interferon, the viral infection may induce the activation of host genes for viral resistance, resulting in the enhanced viral resistance of hosts. Genetic analysis of virus functions may provide more important information on the activation of host genes.
Reconstitution of DNA viruses possessing DNA as the genomic nucleic acid has been performed for some time, and can be carried out by the introduction of the purified genome itself, such as SV40, into monkey cells [J. Exp. Cell Res., 43, 415-425 (1983)].
Reconstitution of RNA viruses containing an RNA genome has been preceded by positive strand RNA viruses since genomic RNAs also function as mRNA. For example, in the case of poliovirus, the disseminative capability of the purified genomic RNA itself was already demonstrated in 1959 [Journal of Experimental Medicine, 110, 65-89 (1959)]. Then, it was achieved to reconstitute Semliki forest virus (SFV) by the introduction of cloned cDNAs into host cells utilizing DNA-dependent RNA polymerase activity of host cells [Journal of Virology, 65, 4107-4113 (1991)].
Furthermore, using these viral reconstitution techniques, gene therapy vectors have been developed [Bio/Technology, 11, 916-920 (1993); Nucleic Acids Research, 23, 1495-1501 (1995); Human Gene Therapy, 6, 1161-1167 (1995); Methods in Cell Biology, 43, 43-53 (1994); Methods in Cell Biology, 43, 55-78 (1994)].
However, as described above, in spite of many advantages of Sendai virus to be industrially useful virus, its reconstitution system has not been established, because it is a negative-strand RNA. This is due to tremendous difficulty in reconstituting viral particles via viral cloned cDNAs.
As described above, it has been clearly demonstrated that a mere introduction of RNA from negative-strand RNA virus (vRNA) or its complementary strand RNA (cRNA) into host cells does not support the generation of negative-strand RNA virus. This is absolutely different from the case of positive strand RNA viruses. Although, in Tokkai H4-211377, “methods for preparing cDNAs corresponding to negative strand RNA viral genome and infectious negative strand RNA virus” are disclosed, the entire experiments of said documents described in “EMBO. J., 9, 379-384 (1990) were later found to be not reproducible, so that the authors themselves had to withdraw all the article contents [see EMBO J., 10, 3558 (1991)]. Therefore, it is obvious that techniques described in Tokkai H4-211377 do not correspond to the related art of the present invention.
Reconstitution systems of negative strand RNA viruses were reported for influenza virus [Annu. Rev. Microbiol., 47, 765-790 (1993); Curr. Opin. Genet . Dev., 2, 77-81 (1992)]. Influenza virus is a negative strand RNA virus having an eight-segmented genome. According to these literatures, a foreign gene was first inserted into the cDNA of one of said genome segments, and then RNA transcribed from the cDNA of all eight segments containing the foreign gene was assembled with the virus-derived NP protein to form RNP. Then, the virus reconstitution was achieved by providing host cells with these RNPs and an RNA-dependent RNA polymerase. Thereafter, the reconstitution of negative single-stranded RNA virus from cDNA was reported for rabies virus belonging to the rhabdovirus family [J. Virol., 68, 713-719 (1994)].
Therefore, although techniques for reconstituting negative strand viruses have become fundamentally known to the public, in the case of Sendai virus, the direct application of these techniques did not support the viral reconstitution. Also, the reconstitution of viral particles reported on the rhabdovirus was confirmed only by the expression of marker genes, RT-PCR, etc. Furthermore, the yield was not satisfactory for practical applications. Besides, in order to provide factors required for the viral reconstitution within host cells, helper viruses such as wild type viruses, recombinant vaccinia virus, etc. were conventionally introduced to host cells together with nucleic acids of the virus to be reconstituted. Accordingly, difficulties in separating the reconstituted desired virus from these harmful viruses were posing a difficult problem.